Method of evaluating an effect of a substance applied to the skin of a mammal

ABSTRACT

A method for evaluating an effect of a substance when applied to mammalian skin is provided. The method comprises the steps of: (i) assessing an immune response of the skin of at least one mammal to an antigen, to which the mammal has been sensitized, in order to obtain data; (ii) applying the substance to the skin; (iii) assessing the immune response of the skin to the antigen following the application of the substance to the skin to obtain further data; and (iv) using the data obtained in steps (i) and (iii) to evaluate the effect of the substance.

TECHNICAL FIELD

The present invention relates to a method for evaluating an effect of asubstance on the immune response of the skin of a mammal. The method maybe used to provide information which can be shown on commercialpreparations of substances so that consumers are better able to make acomparison between such substances. The invention also relates to thesubstance evaluated by the method.

BACKGROUND ART

Exposure to UV radiation can cause erythema (sunburn), the formation ofDNX pyrimidine dimers, premature ageing and dermal connective tissuechanges. It is common to evaluate the protection from erythema providedby a sunscreen preparation by determining its sunscreen protectionfactor (SPF). The practice, of determining SPF is useful not only inproviding information as to the effectiveness of different sunscreenpreparations in protecting against erythema, but also in thedetermination of a minimum level of protection which must be met beforea given sunscreen preparation can be marketed commercially.

Studies have shown that exposure to ultraviolet radiation can alsoprofoundly impair the cutaneous immunity of mammals and it is widelybelieved that ultraviolet radiation induced immunosuppressioncontributes significantly to cutaneous carcinogenesis.

The effects of UV radiation on the immune system include reduced contacthypersensitivity (CHS) and delayed hypersensitivity reactions, systemicimmunosuppression in mice, and tolerance of tumours in mice orepicutaneous antigens in humans Noonan 1981, Hersey 1983, Ullrich 1986,Cestari 1995, Cooper 1992!. In humans, such alterations in immunity canoccur even with small, suberythemal doses of UV.

Where cutaneous immunity is markedly affected by long-term, systemicimmunosuppressive medications, as is seen in transplant recipients, theincidence of non-melanoma skin cancer is ten to one hundred-fold higherthan in age-matched, immunologically competent controls London 1995,Espana 1995!.

It is, therefore, important that human beings who are exposed to evensmall doses of UV radiation be protected not only from the erythemal andmutagenic effects of sunlight, but also from its immunosuppressiveeffects. While sunscreens can decrease the formation of pyrimidine DNAdimers Freeman 1988!, reduce the incidence of pre-malignant solarkeratoses Thompson 1993, Naylor 1995!, can delay and even prevent UVtumorigenesis in mice Kligman 1980!, and may help reduce the incidenceof skin cancer in humans, if they fail to protect the immune system thenindividuals using high SPF sunscreens who tend to stay in the sun forextended periods may become severely immunosuppressed and have anincreased risk of skin malignancy.

Recently, Whitmore et al 1995! found that a high SPF sunscreencompletely prevented UV suppression of induction of primary contactsensitisation to dinitrochloroberizene (DNCB). Work in mice suggeststhat UV immunoprotection is more dependent on broad spectrum cover thanSPF Bestak 1995!, since the erythemal and immunosuppressive spectra areprobably different.

Previous studies of UV suppression of CHS have used induction of primaryCHS to antigens such as DNCB. However, since induction of primary CHSrequires exposure to an antigen for the first time the immune responseto the antigen cannot be anticipated and it is possible the exposure mayelicit a severe response which is, of course, undesirable.

Induction of primary CHS to an antigen also necessarily means that theresultant immune response may only be evaluated once in a given subject.Accordingly, in order to evaluate the ability of a substance to suppressa skin immune response or to evaluate the protection provided by thesubstance against suppression of the immune response by ultravioletradiation using induction of primary CHS, a large number of differentindividuals are required. This is also undesirable and is compounded bythe need to use different groups of individuals each time a substance isevaluated.

With the increasing availability of high SPF sunscreens, such as SPF 30,which encourages individuals to stay in the sun for substantial periodsof time, there is a need for a reliable and relatively convenient methodfor evaluating the amount of immunoprotection afforded by commonly usedsunscreen ingredients and sunscreens in general. Similarly, there is aneed for a suitable and effective method for evaluating whether thecutaneous immunity of an individual is impaired by a substance whenapplied to the individual's skin.

DISCLOSURE OF INVENTION

In a first aspect of the present invention there is provided a method ofevaluating an effect of a substance when applied to mammalian skin,comprising the steps of:

(i) assessing an immune response of the skin of at least one mammal toan antigen, to which the mammal has been sensitised, in order to obtaindata;

(ii) applying the substance to the skin;

(iii) assessing the immune response of the skin to the antigen followingthe application of the substance to obtain further data; and

(iv) using the data obtained in steps (i) and (iii) to determineinformation to evaluate the effect of the substance.

In a second aspect of the invention there is provided the substanceevaluated by the method.

In a third aspect of the invention there is a method of providing aproduct comprising an evaluated substance for topical application tomammalian skin, involving:

(a) evaluating the substance with the use of information obtained by thesteps of:

(i) assessing an immune response of the skin of at least one mammal toan antigen, to which the mammal has been sensitised, to obtain data;

(ii) applying the substance to the skin;

(iii) assessing the immune response of the skin to the antigen followingthe application of the substance to obtain further data; and

(iv) using the data obtained in steps (i) and (iii) to determine theinformation.

Typically, the product will consist of a container such as a jar,bottle, vial, tube or sachet containing the evaluated substance. Theproduct may also comprise packaging that holds or displays thecontainer. Any conventionally known packaging may be used for thispurpose.

If desired, the information can be provided so that the result of theevaluation is shown with the product. Indeed, the information can bemarked directly on the container or packaging, or for instance, beprovided by means of a label, tag or the like attached to the containeror packaging.

The evaluating may comprise determining whether the substance suppressesor enhances the immune response of the skin to the antigen and the levelof suppression or enhancement of the immune response resulting from thecontact of the skin with the substance.

Alternatively, the evaluating may comprise determining whether thesubstance protects the immune response of the skin to the antigen fromultraviolet radiation. In this instance, the method can be used tomeasure the protection provided by the substance to the immune responseof the skin from the ultraviolet radiation. In order to determine ormeasure such protection the skin is exposed to ultraviolet radiation inthe presence of the substance.

The skin may also be exposed to ultraviolet radiation prior to theapplication of the substance. Usually, the minimum dose of ultravioletradiation which suppresses the immune response of the skin in theabsence of the substance and the minimum dose of ultraviolet radiationgiven to the skin in the presence of the substance which suppresses theimmune response will be determined.

This data can be used to calculate an immune protection factor (IPF) forthe substance. However, if desired, other data provided by the methodmay be used to calculate the IPF. Similarly, data obtained whilecarrying out the method can be used to calculate an immune suppressionfactor (ISF) or immune enhancement factor (IEF) in those instances wherethe substance is known or found to suppress or enhance the cutaneousimmunity of the skin.

By evaluating such effects the commercial worth of the substance may beenhanced since consumers will generally prefer substances which havebeen shown to have limited deleterious effects, or which have been shownto have a desired characteristic or a desired level of suchcharacteristic (e.g., a desired IPF value).

The antigen can be a metal, a polypeptide such as purified proteinderivative of tuberculin, or other material. Preferably, the metal isnickel or chrome. Most preferably, the antigen is nickel.

In order to evaluate the immune responses of the skin the antigen may beapplied to the surface of the skin or be injected into the skin.Preferably, patches containing a predetermined dose of the antigen areused to apply the antigen.

Typically, the mammalian skin will be the skin of one or more humanbeings. Since the mammal or mammals have previously been sensitised tothe antigen it will be understood that the antigen is a recall antigen.

By using a recall antigen a mammal can be exposed to the antigen anynumber of times and a consistent immune response obtained on eachchallenge for a given amount of the antigen. Suppression of the immuneresponse to the antigen or protection against ultraviolet radiationprovided to the immune response by different substances can also beevaluated on one mammal at the some time. In addition, the method can beused on the same mammal on different occasions. This is particularlyadvantageous when the mammal is a human being as it avoids the necessityto recruit new people each time a substance is to be evaluated using themethod.

Furthermore, the method allows preliminary testing of the immuneresponse to the antigen so that those with a desired level of responsecan be selected for studies using the method of the invention, and thosehaving low or high responses can be excluded. Moreover, the undesirableuse of potentially carcinogenic compounds such as DNCB on people andethical problems associated with such use can be avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the immunosuppression in unprotected human skinfollowing exposure to UV radiation compared to skin protected bydifferent sunscreen lotions. The immune response was evaluated using anerythema spectrometer; and

FIG. 2 shows the results when a clinical scoring method was used toevaluate the immune response instead of the erythema spectrometer.

MODES FOR CARRYING OUT THE INVENTION

Substances that may be evaluated with the use of the method includesunscreen, cosmetic and pharmaceutical preparations. The preparationsmay be in the form of, for instance, a lotion, cream, gel or ointment.It will further be appreciated that an ingredient or combination ofingredients for possible use in providing such preparations may also beevaluated by the method. In this instance, the ingredient or ingredientscan be mixed with a carrier for the purpose of assisting in applying thesubstance to the skin at a desired concentration, wherein the carrierhas only a minor or negligent effect on the immune response of the skinto the antigen.

A sample taken from a batch of the substance or alternatively, a sampleobtained or prepared separately from the batch to be evaluated can beused in the method. Once evaluated, the batch of the substance can beutilised in the preparation of products marked with or otherwise showingthe result of the evaluation for sale or distribution to consumers.

1.0 EXPERIMENTAL

To evaluate the use of a recall antigen in determining the protectionprovided by a sunscreen against the immunosuppressive effect of UVradiation nickel was selected as it is the most common contact antigento which the general population has been sensitised. It has beenreported that up to 10% of women Peltonen L, 1979! and 4% of men Meijer,1995! develop allergic contact dermatitis when exposed to nickel, mostcommonly in response to contact with earrings, watchbands and otherjewellery.

For the study, people with symptoms suggestive of nickel allergy wererecruited from the general population The individuals were otherwiseimmunologically normal, and had not experienced sun exposure of theirbacks in the four weeks preceding the study. Nickel allergy wasconfirmed in each individual by patch testing with variousconcentrations of nickel sulphate.

1.1 PATCH TESTING

To confirm nickel allergy a number of preparations containing nickelsulphate in a petrolatum base (Trolab Herman, Germany) were placed in 9mm diameter Finn chambers, and the patches taped to the mid to upperback. Each volunteer was initially patch tested with 5 concentrations ofnickel (0.125%, 0.25%, 0.5%, 1% and 2.5%) in order to determine whichconcentration elicited the minimum reaction needed for the study in eachvolunteer. In this way, more severe, vesicular reactions were able to beavoided during subsequent patch testing.

The patches were removed after 48 hours and the response assessed 30minutes later and then 24 hours later by means of a clinical scoringsystem (see Table 1). A positive nickel reaction consisted of erythemawith induration at the site of the patch test. Only volunteers whoproved allergic to nickel and displayed a confluent nickel reaction(score of 3 or more), were used in the study.

                  TABLE 1    ______________________________________    Clinical Scoring System for Assessing Nickel CHS Reactions    SCORE      OBSERVATION    ______________________________________    0          No reaction    1          Isolated vesicles/pustules    2          Limited, non-confluent induration. No vesicles    3          Mild, confluent induration. No vesicles    4          Moderate, confluent induration. No vesicles    5          Vesicles over <25% of the patch area    6          Vesicles over 25-50% of the patch area    7          Vesicles over 50-75% of the patch area    8          Vesicles covering the entire patch area    9          Vesicular reaction spreading beyond the patch               area    10         Bullous reaction    ______________________________________

1.2 SUNSCREENS

Three different sunscreen lotions and an oil-in-water base lotion commonto each sunscreen were used in the study. The three sunscreens contained5% cinnamate, 3.5% cinnamate with 2.0% oxy benzone, and 3.0% cinnamatewith 2.05% zinc oxide, respectively.

1.3 DETERMINATION OF THE SUN PROTECTION FACTOR (SPF) OF THE SUNSCREENS

The SPF of each sunscreen and the base lotion was determined in vivo onthe backs of 5 human volunteers. The UV source was the same fluorescentlamp array used in the irradiation protocol described below, and thelotions were applied at a concentration of 2 mg/cm², 15 minutes prior toirradiation. The area of skin to be irradiated was divided intosegments, to assist in the application of the three different sunscreensand their base lotion to different areas of the volunteers' skin. Alllotions were removed with soap and water at the completion of theirradiation. The total contact time of each sunscreen was less then 25minutes.

The minimum erythemal dose (MED) was determined as the minimum dose atwhich clearly demarcated erythema was observed, and the SPF wascalculated as the ratio of the MEDs of sunscreen-treated and unprotectedskin. The cinnamate only and cinnamate+oxybenzone lotions had an averageSPF of 10, while the cinnamate+zinc oxide lotion was SPF 9.5 and thebase lotion SPF 1.

1.4 IRRADIATION

To assess the effect of UV radiation on the immune response to nickel aUV source comprising an alternating array of 2 UVB (20W Philips TL12)and 6 UVA (20W NEC T10) fluorescent lamps was used. The lamps werefiltered with 0.5 mm cellulose triacetate film (Eastman Kodak,Rochester, N.Y.), to remove any UVC (<290 nm) and to attenuate the lowerwavelength UVB emission so that the spectrum more closely approximatedsunlight. The integrated irradiance of the lamps at the skin surface was0.3245 mW/cm² UV3 and 4 mMW/cm² UVA, determined daily with an IL1350broadband radiometer using SED 038 (UVA) and SED 240 (UVB) detectorscalibrated against the source (CSIRO, Sydney, Australia).

Using this apparatus, the minimum erythemal dose (MED) of the skin ofthe mid to upper back was determined for each nickel sensitive subjectas the lowest dose of UVB at which clearly demarcated erythema was seen.

The skin of the mid-back was then irradiated through a 4 cm×6 cmtemplate with a suberythemal dose of UVB (97.5±2.5 mJ/cm²) andaccompanying UVA (1.23 J/cm²) radiation daily for 5 consecutive days.All individuals (numbering 16) received the same amount of UV radiationregardless of skin type and different 2 cu² areas of the template wereselected in each individual for the daily application of each respectivesunscreen and base lotion prior to irradiation.

After the fifth and final irradiation, the nickel patches were appliedto the back: one patch was placed in each of the four segments used totest the sunscreens and base lotion, one patch tested unprotectedirradiated skin, and two control patches were placed on adjacent,unirradiated skin with and without sunscreens. The patches were left inplace for 48 hours, and the CHS response elicited was assessed bothclinically and with a reflectance spectrometer (Diastron, UK) 24 hoursafter their removal.

All readings with the reflectance spectrometer were taken in triplicatewith subjects resting in the prone position, and an average readingcalculated for each site. There was reasonable correlation betweenclinical scoring of the nickel reactions and objective assessment usingthe erythema spectrometer.

None of the individuals who participated in the study sufferedsignificant adverse effects from the nickel patch testing, and all ofthe eligible volunteers completed the study. However, severalindividuals were excluded from the results because of insufficientnickel reaction at the unirradiated control site (i.e., due to a scoreof less than 3, meaning lack of confluent induration), despite apreviously strong reaction to the initial patch test.

1.5 EVALUATION OF PROTECTION AGAINST IMMUNE RESPONSE SUPPRESSION BY UVRADIATION

An erythema index (EI) was calculated as the difference between theerythema reading at the nickel test site and the erythema reading ofadjacent skin. UV immunosuppression was deterred by comparing the nickelinduced EI at irradiated test sites to unirradiated test sites.

Immunosuppression in unprotected, irradiated skin was not found to varywith either age or MED. When the nickel reactions of unprotected,irradiated skin were compared with those of unirradiated skin there was,on average, immunosuppression of 37% as determined using the erythemameter and 25% using clinical scores as shown in FIGS. 1 and 2respectively. In almost all cases, this attenuation of the nickelreaction was clearly visible and palpable.

The figures also show that the application of the cinnamate onlysunscreen did not prevent significant immunosuppression from occurringsince 18.5% immunosuppression was determined using the erythemaspectrometer while 12.1% immunosuppression was observed using clinicalscores. In contrast, the sunscreen containing both cinnamate andoxybenzone protected against significant immunosuppression. In thisinstance, there was only a 9.1% reduction in the mean erythema index anda 5.3% reduction in the mean clinical score of sites irradiated afterapplication of the sunscreen. The sunscreen containing cinnamate andzinc oxide also prevented significant immunosuppression as indicated bya 10.0% reduction in the mean erythema index and a 7.3% reduction in themean clinical score. Application of the base lotion prior to irradiationhad no effect on either the erythema index or the clinical score.

Each of the three sunscreens and their base lotion was also tested forany effect in the absence of UV. None of the lotions had a significanteffect on the nickel CHS reaction in the absence of UV.

The results show that nickel patch testing is a clinically feasiblemeans of quantitatively assessing UV immunosuppression in humansubjects. The results also show that significant UV immunosuppression ofthe nickel CHS response can be induced in humans with a low dose 5-dayUV protocol, and that even with low doses of UV radiation,immunosuppression is not completely prevented by a relatively high SPFbut narrow spectrum sunscreen (cinnamate).

Rather than inducing primary allergic sensitisation in human subjects,the above model uses CHS to a recall antigen. Individuals can thereforebe tested on more than one occasion and each individual can be used totest several sunscreens. Recent studies of the reproducibility of nickelpatch testing suggest that nickel patch testing on the back gives highlyreproducible results both clinically and with an erythema meter in up to95% of tests Memon 1996!.

EXAMPLE 1

The use of a recall antigen to determine the level of protection againstUV radiation able to be provided to the immune system of a human beingby a sunscreen is described below.

Different 1 cm² areas of the skin of the upper back of an individualwere irradiated with either 0, 50, 70, 90, 110, 130, 150 or 170 mJ/cm²UVB radiation using the UV source described above. Accordingly, the UVBdosages were accompanied by a UVA radiation component which increasedsubstantially proportionately with each UVB dosage. The different areaswere selected using a template laid over the individual's back.

So that at least one of the doses of UVB radiation causedimmunosuppression in the skin of the individual the different areas wereirradiated on five consecutive days at intervals of about one day for aperiod sufficient for each respective area to receive its allocated UVradiation dosage. Each respective area received substantially the samedosage on each exposure to the UV source.

After the final irradiation nickel patches, dosed with the sate amountof 0.5% nickel sulphate, were positioned on each area of the skin for aperiod of 48 hours. The contact hypersensitivity response elicited bythe nickel was then assessed clinically 24 hours after the removal ofthe patches as described above.

The minimum immunosuppressive dose (MID) of UVB found to suppress theimmune response was 70 mJ/cm² UVB.

A 2 mg/cm² amount of the sunscreen was then applied to different 1 cm²areas of the skin of the upper back of the individual 15 minutes priorto irradiation with a multiple of the MID determined above, namely 70,140, 210 or 280 mJ/cm² UVB and using the same UV source.

A further 1 cm² area of the skin of the upper back was irradiated with70 mJ/cm² UVB and accompanying UVA but in the absence of the sunscreenas a negative control. For the positive control another area of the skinto which the sunscreen was not applied and which was not irradiated wasused.

Subsequently, the sunscreens were removed from the skin by washing withsoap and water, and each area of the skin was patch tested with nickelusing the same protocol when determining the MID, and the immuneresponse to the nickel again measured 24 hours after the removal of thepatches.

The minimum dose of UVB radiation which caused suppression of the immuneresponse in the presence of the sunscreen was then determined.

The suppression of the immune response to the nickel was evaluated byfirstly determining the difference in the immune response between thepositive control and the negative control to provide a negative controlerythema index (EI). The difference in the immune response between eacharea irradiated to that obtained for the positive control was thendetermined to provide a test erythema index for each area. Sosignificant difference between a test erythema index and the negativecontrol erythema index indicated that the immune response to the nickelhad been suppressed by the corresponding dosage of UVB radiation,whereas a significantly lower test erythema index compared to thenegative control erythema index indicated that suppression of the immuneresponse had not occurred at that UVB radiation dosage.

In the present example, the immune response of the area of the skinirradiated with 210 mJ/cm² UVB was substantially the same as thatobserved for the area of the skin used as the positive control.Accordingly, no suppression of the immune response was observed at thisdosage of UVB radiation. However, the immune response of the area of theskin irradiated with 280 mJ/cm² UVB and that obtained for the negativecontrol was substantially the same indicating that suppression of theimmune response had occurred.

Hence, the minimum dose of UVB radiation which caused suppression of theimmune response to the nickel in the presence of the substance was 280mJ/cm². The immune protection factors (IPF) of the sunscreen could thenbe calculated by dividing this value by the MID. Using this method, theIPF of the sunscreen is 280/70 which equals 4.

The IPF, therefore, is a measure of the protection provided to theindividual by the sunscreen against suppression of the individual'simmune response by exposure to UVB radiation. Accordingly, a sunscreenwith a higher IPF would provide greater protection against suppressionof the immune response than provided by the sunscreen used in thepresent example.

If desired, the IPF may be calculated in other ways, such as bysubtracting the MID from the minimum dose of UV radiation which causeddepression of the immune response in the presence of the substance. Inthis instance, the IPF would be 280-70 which equals 210. Moreover, whilein the present example the IPF of a sunscreen was determined, the methodmay also be used to evaluate the IPF's of individual components of thesunscreen preparation.

Furthermore, while it is preferable that a UV source is used which emitsboth MUV and UVB radiation, a source emitting only UVA or UVB radiationcan be used in a method of the invention.

If desired, increasing dosages of an amount of UV which is known not tosuppress the immune response of the skin to the nickel may be used todetermine the minimum dosage of UV which causes suppression of theimmune response in the presence of the sunscreen. Accordingly, in thisinstance, it is not necessary to determine the MID and an IPF can becalculated using the dosage known not to cause immunosuppression of theskin.

EXAMPLE 2

A method for determining whether a cosmetic cream or other preparationcauses suppression or enhancement of the immune response of the skin ofa human being when the substance is applied to the skin is describedbelow.

A 1 cm² test area of the skin of the mid-back of an individual is coatedwith a 2 mg/cm² amount of the substance for a period of 3 hours on eachof five consecutive days.

After removal of the substance on the final day a nickel patch dosedwith 0.5% nickel sulphate is applied to the test area. A further nickelpatch containing the same dosage is applied to an area of the skin ofthe upper back as a positive control.

At the end of a 48 hour period the nickel patches are removed and theimmune response of the skin to the nickel is assessed at the test areaand the positive control after a further 24 hour period using theclinical scoring method used in Example 1.

In order to evaluate whether the substance suppressed the cutaneousimmunity of the skin the clinical score for the test area is compared tothe clinical score for the positive control. As will be appreciated,suppression of the immune response is indicated if the clinical scorefor the test area is lower than the clinical score for the positivecontrol while enhancement of the immune response is indicated if theclinical score for the test area is higher than the clinical score forthe positive control.

INDUSTRIAL APPLICABILITY

The method of the invention finds application by manufacturers in thedevelopment of preparations for topical application to the skin ofpersons. In particular, the method enables a manufacturer to determinewhether a given preparation or ingredient for a preparation has anydeleterious effects on the immune response of the skin or alternatively,whether the substance is able to protect the immune response from UVradiation. Once the effect on the immune response is known, themanufacturer can then modify the preparation or select other ingredientsto ameliorate any adverse effect or improve the protection of the immuneresponse provided by the substance.

In addition, the invention finds application in research into cutaneouscarcinogenesis resulting from exposure to ultraviolet radiation orotherwise. The information provided by the method also finds use in theprovision of information useful in the marketing of preparations,whether newly developed or previously known.

Although the present invention has been described hereinbefore withreference to several preferred methods, numerous variations andmodifications are possible without departing from the scope of theinvention which is defined in the following claims.

LIST OF REFERENCES CITED:

1. Noonan E P, de Fabo E C, Kripke M L. Suppression of ContactHypersensitivity to UV Radiation and its Relationship to UV InducedSuppression of Tumour Immunity. Photochem Photobiol 1981; 34:683-689.

2. Hersey P, Hasic E, Edwards A, Bradley M, Haran G, McCarthy W H.Immunological Effects of Solarium Exposure. Lancet 1983; 545-548.

3. Ullrich S E, Azizi E, Kripke M L. Suppression of the Induction ofDelayed-Type Hypersensitivity Reactions in Mice by a Single Exposure toUltraviolet Radiation. Photochem Photobiol 1986; 6:633-638.

4. Cestari T F, Kripke M L, Baptista P L, Bakos L, Bucana C D.Ultraviolet Radiation Decreases the Granulomatous Response to Leprominin Humans. J Invest Dermatol 1995; 103:8-13.

5. Cooper K D, Oberhelman L, Hamilton T A, Baadsgaard O, Terhune M,LeVee G et al. UV Exposure Reduces Immunisation Rates and PromotesTolerance to Epicutaneous Antigens in Humans: Relationship to Dose,CDla-DR+ Epidermal Macrophage Induction, and Langerhans Cell Depletion.Proc Natl Acad Sci 1992; 89:8497-8501.

6. London L J, Farmery S M, Will E J, Davison P M, Lodge J P A. Risk ofNeoplasia in Renal Transplant Recipients. Lancet 1995: 346;403-406.

7. Espana A, Redondo P, Fernandez A L, Zabala M, Herreros J. Llorens Ret al. Skin Cancer in Heart Transplant Patients. J Am Acad Dermatol1995; 32:458-466.

8. Freeman S E, Ley R D, Ley K D. Sunscreen Protection AgainstUV-Induced Pyrimidine Dimers of DNA in Human Skin in situ. Photodermatol1988, 5:243-247.

9. Thompson S C, Jolley D, Marks R. Reduction of Solar Keratoses byRegular Sunscreen Use. N Engl J Med 1993; 329:1147-51.

10. Naylor M F, Boyd A, Smith D W, Cameron G S, Hubbard D, Neldner K H.High Sun Protection Factor Sunscreens in the Suppression of ActinicNeoplasia. Arch Dermatol 1995; 131:170-175.

11. Kilgman L E, Akin P J, Kligman A M. Sunscreens Prevent UltravioletCarcinogenesis. J Am Acad Dermatol 1980; 3:30-35.

12. Whitmore S E, Morison W L. Prevention of UVB-InducedImmunosuppression in Humans by a High Sun Protection Factor Sunscreen.Arch Dermatol 1995; 131:1128-1133.

13. Bestak R, Barnetson R StC, Nearn M R, Halliday G M. SunscreenProtection of Contact Hypersensitivity Responses from ChronicSolar-Simulated Ultraviolet Irradiation Correlates with the AbsorptionSpectrum of the Sunscreen. J Invest Dermatol 1995; 105:345-351.

14. Peltonen L. Nickel Sensitivity in the General Population. ContactDermatitis 1979; 5:27-32.

15. Meijer C, Bredberg M, Fischer T, Widstrom L. Ear Piercing, andNickel and Cobalt Sensitisation, in 520 Young Swedish Men DoingCompulsory Military Service. Contact Dermatitis. 1995; 32:147-149.

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We claim:
 1. A method of evaluating an effect of a substance whenapplied to mammalian skin, comprising the steps of:(i) assessing animmune response of the skin of at least one mammal to an antigen, towhich the mammal has been sensitised, in order to obtain data; (ii)applying the substance to the skin; (iii) assessing the immune responseof the skin to the antigen following the application of the substance toobtain further data; and (iv) using the data obtained in steps (i) and(iii) to determine information to evaluate the effect of the substance.2. A method according to claim 1 wherein the method further comprisesthe step of substantially removing the substance from the skin prior tostep (iii).
 3. A method according to claim 1 wherein a batch of thesubstance is to be evaluated and a sample from the batch or a sample ofthe substance obtained separately from the batch is used in theevaluation.
 4. A method according to claim 1, wherein the evaluatingcomprises determining whether the substance suppresses, enhances orprotects the immune response of the skin to the antigen.
 5. A methodaccording to claim 1 wherein the substance is a sunscreen, cosmetic orpharmaceutical preparation.
 6. A method according to claim 4, whereinstep (ii) further comprises exposing the skin to ultraviolet radiationand the evaluating comprises determining the level of protectionprovided by the substance to the immune response of the skin from theultraviolet radiation.
 7. A method according to claim 6 wherein the skinis exposed to a selected range of dosages of the ultraviolet radiationthat includes dosages able to suppress the immune response of the skinto the antigen in the absence of the application of the substance to theskin, and step (iii) involves assessing the immune response of the skinat each of the dosages.
 8. A method according to claim 7 wherein each ofthe dosages of the ultraviolet radiation are applied to the skin over apredetermined number of days.
 9. A method according to claim 7 whereinstep (i) further comprises exposing the skin to an initial range ofdosages of ultraviolet radiation and the immune response of the skin isassessed at each of those dosages, and wherein at least one of theinitial dosages suppresses the immune response.
 10. A method accordingto claim 9 wherein the initial and the selected range of dosages ofultraviolet radiation are applied to the skin over a predeterminednumber of days, respectively.
 11. A method according to claim 10 whereinobtaining the data in steps (i) and (iii) comprises determining minimumdosages of the ultraviolet radiation that suppress the immune responseof the skin to the antigen.
 12. A method according to claim 1, whereinthe antigen is selected from the group consisting of a metal and apolypeptide.
 13. A method according to claim 1, wherein the antigen isnickel or chrome.
 14. A method according to claim 1, wherein the antigenis applied to the surface of the skin or is injected into the skin toevaluate the immune response of the skin in steps (i) and (iii).